The specificity of the T cell recognition process is determined by the nature of interactions between the T cell receptor (TCR) and its ligand, peptide-MHC (pMHC) complex. For MHC class I restricted responses, the coreceptor CD8 provides co-stimulatory signals and has the potential to modulate the T cell response during thymic development and T cell activation. However, the precise role played by CD8 during TCR binding to pMHC (peptide-MHC) ligands is not clearly defined. It is not clear whether CD8 has a direct influence on TCR-pMHC binding, or its effect is purely due to enhanced signal transduction, or both. It has been proposed that CD8 might interact only with TCR-pMHC complexes that are relatively long-lived. Alternatively, CD8 might interact directly during TCR-pMHC complex formation, and consequently modulate TCR-pMHC interactions. It is reasonable to believe that co-stimulatory signals would be most effective and advantageous in enhancing low affinity TCR-pMHC interactions as in the case of weak agonist or positive selecting ligands. To address these issues, we propose to use a novel BIAcore binding assay to quantitate TCR-pMHC, CD8-MHC, and CD8-MHC-TCR trimolecular interactions on membranes expressing intact TCR and coreceptors. This technique will allow us to study the interactions between these molecules under conditions where TCR and co-receptors are expressed in their native conformations and environment. The specific aims of the project are: (1) to measure the binding kinetics and affinity of the interactions between membrane associated intact TCR-CD3 (mTCR) and peptide-MHC(pMHC) complexes; (2) to study the influence of CD8 on ligand specific binding of mTCR to pMHC complexes; (3) to define the thermodynamics of TCR binding to pMHC complex and the influence of coreceptor CD8; and (4) to study the influence of glycosylation on the binding of TCR and CD8. The kinetic and thermodynamic characterization of CD8 molecule as a modulator of TCR-pMHC interactions can serve as a basis for understanding immunomodulation of T cell responses, particularly against pathogens that elicit low avidity cytotoxic T lymphocyte (CTL).